DESCRIPTION: This is an amended proposal to understand the structural basis of G protein-coupled receptor activation. Specifically proposed is the analysis of constitutive activity exhibited by the yeast alpha-pheromone receptor, and subsequently the mammalian adenosine receptor, in yeast using a novel, lacZ-based reporter assay. The first aim outlines strategies to determine which residues, when mutated, cause constitutive activity. Both directed (TM5 and 6) and random mutagenesis will be used, with the idea that the mapping will provide the material for subsequently proposed assays of ligand binding and structural changes. The second aim addresses quantification of constitutive activity to provide a relative ranking of activity among the mutants and an assessment of ligand binding. The third aim deals with the analysis of receptor structure using a method that examines spatial proximity of selected residues. The method is based on the insertion of cysteine residues to form intramolecular disulfide bonds. While the first three aims deal with the alpha-pheromone receptor, the fourth aim represents an extension of the developed techniques to the mammalian adenosine receptor in order to determine common principles in GPCR activation.